Circular nucleic acid vectors, and methods for making and using the same

ABSTRACT

Circular nucleic acid vectors that provide for persistently high levels of protein expression are provided. The circular vectors of the subject invention are characterized by being devoid of expression-silencing bacterial sequences, where in many embodiments the subject vectors include a unidirectional site-specific recombination product hybrid sequence in addition to an expression cassette. Also provided are methods of using the subject vectors for introduction of a nucleic acid, e.g., an expression cassette, into a target cell, as well as preparations for use in practicing such methods. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications. Also provided is a highly efficient and readily scalable method for producing the vectors employed in the subject methods, as well as reagents and kits/systems for practicing the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit and is a Divisional of application Ser.No. 14/480,420 filed Sep. 8, 2014, which is a Divisional of applicationSer. No. 11/398,490 filed Apr. 4, 2006, now U.S. Pat. No. 8,828,726,issued Sep. 9, 2014, which is a Divisional of application Ser. No.10/652,729 filed Aug. 28, 2003, now U.S. Pat. No. 7,897,380, issued Mar.1, 2011, which claims benefit of U.S. Provisional Patent ApplicationSer. No. 60/407,344 filed Aug. 29, 2002 and U.S. Provisional PatentApplication Ser. No. 60/463,672 filed Apr. 16, 2003; the disclosures ofwhich are herein incorporated by reference.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract HL064274awarded by the National Institutes of Health. The Government has certainrights in the invention.

INTRODUCTION Field of the Invention

The field of this invention is molecular biology, particularlytransformation and specifically vectors employed in transformation.

Background of the Invention

The introduction of an exogenous nucleic acid sequence (e.g., DNA) intoa cell, a process known as “transformation,” plays a major role in avariety of biotechnology and related applications, including research,synthetic and therapeutic applications. Research applications in whichtransformation plays a critical role include the production oftransgenic cells and animals. Synthetic applications in whichtransformation plays a critical role include the production of peptidesand proteins, as well as therapeutic RNAs, such as interference RNA orribozymes. Therapeutic applications in which transformation plays a keyrole include gene therapy applications. Because of the prevalent roletransformation plays in the above and other applications, a variety ofdifferent transformation protocols have been developed.

In many transformation applications, it is desirable to introduce theexogenous DNA in a manner such that it provides for long-term expressionof the protein encoded by the exogenous DNA. Long-term expression ofexogenous DNA is to primarily achieved through incorporation of theexogenous DNA into a target cell's genome. One means of providing forgenome integration is to employ a vector that is capable of homologousrecombination. Techniques that rely on homologous recombination can bedisadvantageous in that the necessary homologies may not always exist;the recombination events may be slow; etc. As such, homologousrecombination based protocols are not entirely satisfactory.

Accordingly, alternative viral based transformation protocols have beendeveloped, in which a viral vector is employed to introduce exogenousDNA into a cell and then subsequently integrate the introduced DNA intothe target cell's genome. Viral based vectors finding use includeretroviral vectors, e.g., Moloney murine leukemia viral based vectors.Other viral based vectors that find use include adenovirus derivedvectors, HSV derived vectors, sindbis derived vectors, etc. While viralvectors provide for a number of advantages, their use is not optimal inmany situations. Disadvantages associated with viral based vectorsinclude immunogenicity, viral based complications, as well asintegration mediated mutation problems, and the like.

Therefore, there is continued interest in the development of additionalmethods of transforming cells with exogenous nucleic acids to providefor persistent, long-term expression of an encoded protein. Ofparticular interest is the development of a non-viral in vivo nucleicacid transfer protocol and vector that provides for persistent proteinexpression without concomitant genome integration, where the vectorprovides for persistent expression in a manner that is independent ofthe sequence and direction of the of the expression cassette present onthe vector.

RELEVANT LITERATURE

U.S. Patents of interest include U.S. Pat. Nos. 5,985,847 and 5,922,687.Also of interest is WO/11092. Additional references of interest include:Wolff et al., “Direct Gene Transfer Into Mouse Muscle In Vivo,” Science(March 1990) 247: 1465-1468; Hickman et al., “Gene Expression FollowingDirect Injection of DNA Into Liver,” Hum. Gen. Ther. (December 1994)5:1477-1483; and Acsadi et al., “Direct Gene Transfer and ExpressionInto Rat Heart In Vivo,” New Biol. (January 1991) 3:71-81.

SUMMARY OF THE INVENTION

Circular nucleic acid vectors that provide for persistently high levelsof protein expression are provided. The circular vectors of the subjectinvention are characterized by being devoid of expression-silencingbacterial sequences, where in many embodiments the subject vectorsinclude a unidirectional site-specific recombination product sequence inaddition to an expression cassette. Also provided are methods of usingthe subject vectors for introduction of a nucleic acid, e.g., anexpression cassette, into a target cell, as well as preparations for usein practicing such methods. The subject methods and compositions finduse in a variety of different applications, including both research andtherapeutic applications. In addition, methods for making such vectors,as well as reagents and kits/systems for practicing the same, are alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a to 1d . φC31-mediated production of minicircle in E. coli. FIG.1a , Flow chart of φC31 integrase-mediated intramolecular recombinationof pBADφC31.RHB and resulting DNA products cBB and MC.RHB. RSV, RousSarcoma virus long terminal repeat promoter; hAAT, human a1-antitrypsin; bpA, bovine growth factor polyadenylation signal; RHB,RSV.hAAT.bpA expression cassette; Amp, ampicillin resistant gene; BAD,araBAD promoter; araC, araC repressor; attB, bacterial attachment site;attP, phage attahment site; attL, left hybrid sequence; attR, righthybrid sequence; UC, pUC origin of DNA replication. MC, minicircle; cBB,circular bacterial backbone. Restriction sites: B, BamH1; N, Nco I; S,Spe I; and X, Xho I. FIG. 1b , The vector pBAD.φ031.hFIX used forproduction of minicircle expressing human factor IX (hFIX). sApoE, theartificial enhancer/promoter sApoE.HCR.hAAT¹⁷; Int A, Intron A. FIG. 1c, Kinetic analysis of L-arabinose-induced φ031-mediated formation ofMC.RHB. The influence of different bacteria broth conditions on MC.RHBproduction was determined. Re-suspension: 4:1 and 1:1, represents thevolume of overnight bacterial growth versus volume of fresh LB brothcontaining 1% L-(+)-arabinose used to resuspend the bacteria; None: 1%L-(+)-arabinose was added directly to the overnight bacterial growth.Bacterial plasmid DNA was isolated from growth media and purified. Eachlane loaded with 1 μg of BamH1 digested DNA. The 2.1, and 6.0 kb bandsrepresented the linear MC.RHB, and cBB, respectively, while the 4.5 and3.5 kb bands were derived from the un-recombined pBAD.φ031,RHB. FIG. 1d, Determination of the time course of minicircle formation byquantification of DNA bands in the gel of FIG. 1c . The values ofminicircle ratio are presented as the percent of the 2.1 kb linearminicircle band compared to the combination of all 4 bands in each lane.

FIGS. 2a to 2b . Trangene expression profiles. FIG. 2a , The vectorpRSV.hAAT.bpA used for preparing the 3 different forms of DNA. FIG. 2b ,Serum hAAT and hFIX expression. Left panel, serum hAAT from mice thatreceived 20.0 μg of closed circular pRSV.hAAT.bpA (CC), or equivalentmolar amounts of purified expression cassette (1f, 8.2 μg), 2-fragmentDNA (2f, 20.0 μg), or minicircle DNA (MC, 8.5 μg). Right panel, serumhFIX from mice that received 40.0 μg of—unrecombinedplasmid—pBAD.φ031.hFIX (FIG. 1b ) or equal molar amount of minicircle(16.2 μg).

FIGS. 3a to 3b . Southern blot analysis of vector DNA in mouse livers.Liver DNA from mice treated as indicated in the legend of FIG. 2b leftpanel. FIG. 3a , Quantification of vector DNA in mouse livers. 20.0 μgof liver DNA was digested with EcoR1 to release the 1.4 kb hAAT cDNA(FIGS. 1a, and 2a ) and quantified by Phosphalmager. FIG. 3b , Molecularstructure of vector DNA in mouse livers. Twenty μg of liver DNA wasdigested with Bgl II (does not cut in the vector), or Hind III (cutsonce in the vector), and vector expression cassette DNA bands visualizedafter hybridization with a radio-labeled hAAT cDNA probe.

FIGS. 4a to 4c provide a schematic represention of a secondrepresentative minicircle vector preparation protocol.

FIG. 5 provides a representation of a gel that demonstrates the purityof a minicricle preparation produced by the protocol of FIGS. 4a to 4 c.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Circular nucleic acid vectors that provide for persistently high levelsof protein expression are provided. The circular vectors of the subjectinvention are characterized by being devoid of expression-silencingbacterial sequences, where in many embodiments the subject vectorsinclude a unidirectional site-specific recombination product sequence inaddition to an expression cassette. Also provided are methods of usingthe subject vectors for introduction of a nucleic acid, e.g., anexpression cassette, into a target cell, as well as preparations for usein practicing such methods. The subject methods and compositions finduse in a variety of different applications, including both research andtherapeutic applications. In addition, methods for making such vectors,as well as reagents and kits/systems for practicing the same, are alsoprovided.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the cell lines, vectors,methodologies and other invention components that are described in thepublications which might be used in connection with the presentlydescribed invention.

Methods

In the broadest sense, the present invention provides methods ofintroducing an exogenous nucleic acid into at least one cell, i.e., atarget cell. The target cell may be an individual cell, e.g., as may bepresent in an in vitro environment, or present in a multicellularorganism. As such, the subject methods may be in vivo methods, by whichis meant that the exogenous nucleic acid is administered directly to themulticellular organism, or in vitro methods, in which the target cell orcells are removed from the multicellular organism and then contactedwith the exogenous nucleic acid.

In certain embodiments, the present invention provides methods ofintroducing an exogenous nucleic acid into a plurality of the cells of amulticellular organism, i.e., a host, where by “plurality” is oftenmeant at least about 0.1 number %, usually at least about 0.5 number %in certain embodiments.

As specified below, in many in vitro embodiments the subject methodsrely on systemic administration of the vector employed in the subjectmethods, where by systemic administration is meant that the vector isadministered to the host in a manner such that it comes into contactwith more than just a local area or region of the host, where by localarea or region of the host is meant a region that is less than about10%, usually less than about 5% of the total mass of the host. In otherin vitro embodiments, local administration protocols are employed. Whilein the broadest sense the subject methods are methods of introducing anynucleic acid into a host, generally, the exogenous nucleic acid is anexpression cassette that encodes a product, e.g., protein, of interest,as described in greater detail infra.

Minicircle Vector

A feature of the subject invention is that the methods employ a minimalcircular vector, i.e., a minicircle, to deliver the exogenous nucleicacid, hereinafter referred to as “expression cassette” for convenience,to the target cell or cells. The minicircle vector employed in thesubject methods is a double-stranded circular DNA molecule. The sequenceof the minicircle vector employed in the subject methods is such that itprovides for persistent, high level expression of an expression cassetteencoded protein present on the vector in a manner that is at leastsubstantially expression cassette sequence and direction independent.

As summarized directly above, a feature of the subject minicirclevectors is that they provide for persistent expression of the expressioncassette encoded protein present thereon, as opposed to transient orshort-lived expression. By persistent expression is meant that theexpression of encoded product, e.g., protein, is at a detectable levelthat persists for an extended period of time, if not indefinitely,following administration of the subject vector. By extended period oftime is meant at least 1 week, usually at least 2 months and moreusually at least 6 months. By detectable level is meant that theexpression of the encoded product is at a level such that one can detectthe encoded product in target cell, or the mammal comprising the same,e.g., in the serum of the mammal, at a therapeutic concentration. Seee.g., the experimental section, supra. As compared to a control in whichthe pBluescript plasmid vector (Stratagene Corporation, La Jolla,Calif.) is employed, protein expression persists for a period of time ata detectable level that is at least about 2 fold, usually at least about5 fold and more usually at least about 10 fold longer following thesubject methods as compared to a control. An encoded product isconsidered to be at a detectable level if it can be detected usingtechnology and protocols readily available and well known to those ofskill in the art. The experimental section infra provides representativedetectable levels of the human factor IX protein in mouse serum.

Typically, the above-described persistent expression is not only at adetectable level, but at a high level. A minimal vector is considered toprovide for a high level of expression if, after a period of timefollowing its administration, e.g., at least about 28 days, the proteinencoded by the expression cassette of the vector is present at highlevels in the host, e.g., in the target cells, in the serum of the host,etc. Levels of an encoded product are considered “high” for purposes ofthe present application if they are present in amounts such that theyexhibit detectable activity (e.g., have an impact on the phenotype),e.g., therapeutic activity, in the host. Whether or not the expressionlevel of a particular product is high will necessarily vary depending onthe nature of the particular product, but can readily be determined bythose of skill in the art, e.g., by an evaluation of whether expressionof the product is sufficient to exhibit a desired effect on thephenotype of the host, such as an amelioration of a disease symptom,e.g., reducing clotting time, etc. A minicircle vector according to thesubject invention can be tested to see if it provides for the requisitehigh level of protein expression by administering it to a host accordingto the protocols described, infra, and testing for the desiredexpression level, e.g., in the blood or serum where the expressionprotein is secreted from the target cell where it is produced, in atissue lysate of the target cells for non-secreted proteins, and thelike.

The minicircle vectors employed in the subject methods include severalelements that provide for their utility in the subject methods. Thesubject minicircle vectors include at least one restriction endonucleaserecognized site, i.e., a restriction site, which typically serves as acloning site, i.e., a site into which nucleic acid may be inserted. Avariety of restriction sites are known in the art and may be included inthe vector, where such sites include those recognized by the followingrestriction enzymes: HindIII, PstI, SalI, AccI, HincII, XbaI, BamHI,SmaI, XmaI, KpnI, SacI, EcoRI, and the like. In many embodiments, thevector includes a polylinker (also known in the art as a multiplecloning site), i.e., a closely arranged series or array of sitesrecognized by a plurality of different restriction enzymes, such asthose listed above. As such, in many embodiments, the vectors include amultiple cloning site made up of a plurality of restriction sites. Thenumber of restriction sites in the multiple cloning site may vary,ranging anywhere from 2 to 15 or more, usually 2 to 10.

When employed, the minicircle vectors typically include at least onenucleic acid of interest, i.e., a nucleic acid that is to be introducedinto the target cell, e.g., to be expressed as protein in the targetcell, etc., as described in greater detail below, where the nucleic acidis typically present as an expression cassette. The subject vectors mayinclude a wide variety of nucleic acids, where the nucleic acids mayinclude a sequence of bases that is endogenous and/or exogenous to thetarget cell/multicellular organism, where an exogenous sequence is onethat is not present in the target cell while an endogenous sequence isone that pre-exists in the target cell prior to introduction. In anyevent, the nucleic acid of the vector is exogenous to the target cell,since it originates at a source other than the target cell and isintroduced into the cell by the subject methods, as described infra. Thenature of the nucleic acid will vary depending the particular protocolbeing performed. For example, in research applications the exogenousnucleic acid may be a novel gene whose protein product is not wellcharacterized. In such applications, the vector is employed to stablyintroduce the gene into the target cell and observe changes in the cellphenotype in order to characterize the gene. Alternatively, in proteinsynthesis applications, the exogenous nucleic acid encodes a protein ofinterest which is to be produced by the cell. In yet other embodimentswhere the vector is employed, e.g., in gene therapy, the exogenousnucleic acid is a gene having therapeutic activity, i.e., a gene thatencodes a product of therapeutic utility.

A variety of different features may be present in the vector. In manyembodiments, the vector is characterized by the presence of at least onetranscriptionally active gene. By transcriptionally active gene is meanta coding sequence that is capable of being expressed under intracellularconditions, e.g., a coding sequence in combination with any requisiteexpression regulatory elements that are required for expression in theintracellular environment of the target cell into which the vector isintroduced by the subject methods. As such, the transcriptionally activegenes of the subject vectors typically include a stretch of nucleotidesor domain, i.e., expression module or expression cassette, that includesa coding sequence of nucleotides in operational combination, i.e.operably linked, with requisite transcriptional mediation or regulatoryelement(s). Requisite transcriptional mediation elements that may bepresent in the expression module include promoters, enhancers,termination and polyadenylation signal elements, splicing signalelements, and the like.

Preferably, the expression module or expression cassette includestranscription regulatory elements that provide for expression of thegene in a broad host range. A variety of such combinations are known,where specific transcription regulatory elements include: SV40 elements,as described in Dijkema et al., EMBO J. (1985) 4:761; transcriptionregulatory elements derived from the LTR of the Rous sarcoma virus, asdescribed in Gorman et al., Proc. Nat'l Acad. Sci USA (1982) 79:6777;transcription regulatory elements derived from the LTR of humancytomegalovirus (CMV), as described in Boshart et al., Cell (1985)41:521; hsp70 promoters, (Levy-Holtzman, R. and I. Schechter (Biochim.Biophys. Acta (1995) 1263: 96-98) Presnail, J. K. and M. A. Hoy, (Exp.Appl. Acarol. (1994) 18: 301-308)) and the like.

In many embodiments, the at least one transcriptionally active gene ormodule encodes a protein that has therapeutic activity for themulticellular organism, where such proteins include, but are not limitedto: factor VIII, factor IX, β-globin, low-density lipoprotein receptor,adenosine deaminase, purine nucleoside phosphorylase, sphingomyelinase,glucocerebrosidase, cystic fibrosis transmembrane conductance regulator,al-antitrypsin, CD-18, ornithine transcarbamylase, argininosuccinatesynthetase, phenylalanine hydroxylase, branched-chain α-ketoaciddehydrogenase, fumarylacetoacetate hydrolase, glucose 6-phosphatase,α-L-fucosidase, β-glucuronidase, α-L-iduronidase, galactose 1-phosphateuridyltransferase, interleukins, cytokines, small peptides etc, and thelike. The above list of proteins refers to mammalian proteins, and inmany embodiments human proteins, where the nucleotide and amino acidsequences of the above proteins are generally known to those of skill inthe art.

In certain embodiments, the vector also includes at least onetranscriptionally active gene or expression module that functions as aselectable marker. A variety of different genes have been employed asselectable markers, and the particular gene employed in the subjectvectors as a selectable marker is chosen primarily as a matter ofconvenience. Known selectable marker genes include: the thymidine kinasegene, the dihydrofolate reductase gene, the xanthine-guaninephosporibosyl transferase gene, CAD, the adenosine deaminase gene, theasparagine synthetase gene, the antibiotic resistance genes, e.g.,neo^(r) (aminoglycoside phosphotransferase genes), the hygromycin Bphosphotransferase gene, genes whose expression provides for thepresence of a detectable product, either directly or indirectly, e.g.β-galactosidase, GFP, and the like.

An important feature of the subject minicircle vectors employed in thesubject methods is that they do not include bacterial plasmid sequencesthat would cause the vector to provide only transient, as opposed topersistent, expression. Expression is considered to be transient ifexpression is not persistent according to the guidelines provided above.Bacterial sequences that are to be excluded from the subject vectors canreadily be determined by those of skill in the art using the evaluationassays provided in the Experimental section, below.

A feature of certain embodiments of the subject invention is that thevectors further include a product hybrid sequence of a unidirectionalsite-specific recombinase. This product hybrid sequence is the result ofa unidirectional site specific recombinase mediated recombination of tworecombinase substrate sequences, e.g., attB and attP substrate sequencesas they are known in the art, and may be either the attR or attL producthybrid sequence. Typically, the product hybrid sequence ranges in lengthfrom about 10 to about 500 bp. In certain embodiments, the productsequence is a product hybrid sequence of a unidirectional site specificrecombinase that is an integrase, where integrases of interest include,but are not limited to: wild-type phage integrases or mutants thereof,where specific representative integrases of interest include, but arenot limited to: the integrases of ΦC31, R4, TP901-1, A118, ΦFC1 and thelike.

The overall length of the subject minicircle vectors is sufficient toinclude the desired elements as described above, but not so long as toprevent or substantially inhibit to an unacceptable level the ability ofthe vector to enter the target cell upon contact with the cell, e.g.,via system administration to the host comprising the cell. As such, theminicircle vector is generally at least about 0.3 kb long, often atleast about 1.0 kb long, where the vector may be as long as 10 kb orlonger, but in certain embodiments do not exceed this length.

The above-described minicircle vectors may be produced using anyconvenient protocol. An embodiment of how to construct the vectorsemployed in the subject methods is provided, infra.

Vector Administration

The subject methods find use in a variety of applications in which it isdesired to introduce an exogenous nucleic acid sequence into a targetcell, and are particularly of interest where it is desired to express aprotein encoded by an expression cassette in a target cell. As mentionedabove, the subject vectors may be administered by in vitro or in vivoprotocols.

As indicated above, the subject vectors can be used with a variety oftarget cells, where target cells in many embodiments are non-bacterialtarget cells, and often eukaryotic target cells, including, but notlimited to, plant and animal target cells, e.g., insect cells,vertebrate cells, particularly avian cells, e.g., chicken cells, fish,amphibian and reptile cells, mammalian cells, including murine, porcine,ovine, equine, rat, ungulates, dog, cat, monkey, and human cells, andthe like.

In the methods of the subject invention, the vector is introduced intothe target cell. Any convenient protocol may be employed, where theprotocol may provide for in vitro or in vivo introduction of the vectorinto the target cell, depending on the location of the target cell. Forexample, where the target cell is an isolated cell, the vector may beintroduced directly into the cell under cell culture conditionspermissive of viability of the target cell, e.g., by using standardtransformation techniques. Such techniques include, but are notnecessarily limited to: viral infection, transformation, conjugation,protoplast fusion, electroporation, particle gun technology, calciumphosphate precipitation, direct microinjection, viral vector delivery,and the like. The choice of method is generally dependent on the type ofcell being transformed and the circumstances under which thetransformation is taking place (i.e. in vitro, ex vivo, or in vivo). Ageneral discussion of these methods can be found in Ausubel, et al,Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

Alternatively, where the target cell or cells are part of amulticellular organism, the targeting vector may be administered to theorganism or host in a manner such that the targeting construct is ableto enter the target cell(s), e.g., via an in vivo or ex vivo protocol.By “in vivo,” it is meant in the target construct is administered to aliving body of an animal. By “ex vivo” it is meant that cells or organsare modified outside of the body. Such cells or organs are typicallyreturned to a living body. Methods for the administration of nucleicacid constructs are well known in the art. Nucleic acid constructs canbe delivered with cationic lipids (Goddard, et al, Gene Therapy,4:1231-1236, 1997; Gorman, et al, Gene Therapy 4:983-992, 1997;Chadwick, et al, Gene Therapy 4:937-942, 1997; Gokhale, et al, GeneTherapy 4:1289-1299, 1997; Gao, and Huang, Gene Therapy 2:710-722,1995,), using viral vectors (Monahan, et al, Gene Therapy 4:40-49, 1997;Onodera, et al, Blood 91:30-36, 1998,), by uptake of “naked DNA”, andthe like. Techniques well known in the art for the transformation ofcells (see discussion above) can be used for the ex vivo administrationof nucleic acid constructs. The exact formulation, route ofadministration and dosage can be chosen empirically. (See e.g. Fingl etal., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 pl).

The route of administration of the vector to the multicellular organismdepends on several parameters, including: the nature of the vectors thatcarry the system components, the nature of the delivery vehicle, thenature of the multicellular organism, and the like, where a commonfeature of the mode of administration is that it provides for in vivodelivery of the vector components to the target cell(s) via a systemicroute. Of particular interest as systemic routes are vascular routes, bywhich the vector is introduced into the vascular system of the host,e.g., an artery or vein, where intravenous routes of administration areof particular interest in many embodiments.

Any suitable delivery vehicle may be employed, where the deliveryvehicle is typically a pharmaceutical preparation that includes aneffective amount of the vector present in a pharmaceutically acceptablecarrier, diluent and/or adjuvant. In certain embodiments, the vector isadministered in an aqueous delivery vehicle, e.g., a saline solution. Assuch, in many embodiments, the vector is administered intravascularly,e.g., intraarterially or intravenously, employing an aqueous baseddelivery vehicle, e.g., a saline solution.

In many embodiments, the vector is administered to the multicellularorganism in an in vivo manner such that it is introduced into a targetcell of the multicellular organism under conditions sufficient forexpression of the nucleic acid present on the vector to occur. A featureof the subject methods is that they result in persistent expression ofthe nucleic acid present thereon, as opposed to transient expression, asindicated above. By persistent expression is meant that the expressionof nucleic acid at a detectable level persists for an extended period oftime, if not indefinitely, following administration of the subjectvector. By extended period of time is meant at least 1 week, usually atleast 2 months and more usually at least 6 months. By detectable levelis meant that the expression of the nucleic acid is at a level such thatone can detect the encoded protein in the mammal, e.g., in the serum ofthe mammal, at a level of at detectable levels at a therapeuticconcentration. See e.g., the experimental section, supra. As compared toa control in which a pBluescript vector is employed, protein expressionpersists for a period of time that is at least about 2 fold, usually atleast about 5 fold and more usually at least about 10 fold longerfollowing the subject methods as compared to a control.

A feature of many embodiments of the subject methods is that theabove-described persistent expression is achieved without integration ofthe vector DNA into the target cell genome of the host. As such, thevector DNA introduced into the target cells does not integrate into,i.e., insert into, the target cell genome, i.e., one or more chromosomesof the target cell. In other words, the vector DNA introduced by thesubject methods does not fuse with or become covalently attached tochromosomes present in the target cell into which it is introduced bythe subject methods. Accordingly, the vectors are maintained episomally,such that they are episomal vectors that provide for persistentexpression.

The particular dosage of vector that is administered to themulticellular organism in the subject methods varies depending on thenature of vector, the nature of the expression module and gene, thenature of the delivery vehicle and the like. Dosages can readily bedetermined empirically by those of skill in the art. For example, inmice where the vectors are intravenously administered in a salinesolution vehicle, the amount of vector that is administered in manyembodiments typically ranges from about 2 to 100 and usually from about10 to 50 μg/mouse. The subject methods may be used to introduce nucleicacids of various sizes into the a target cell. Generally, the size ofDNA that is inserted into a target cell using the subject methods rangesfrom about 1 to 12 kb, usually from about 3 to 10 kb, and sometimes fromabout 4 to 8 kb.

In in vivo protocols, the subject methods may be employed to introduce anucleic acid into a variety of different target cells. Target cells ofinterest include, but are not limited to: muscle, brain, endothelium,hepatic, and the like. Of particular interest in many embodiments is theuse of the subject methods to introduce a nucleic acid into at least ahepatic cell of the host.

Utility

The subject methods find use in a variety of applications in which theintroduction of a nucleic acid into a target cell is desired.Applications in which the subject vectors and methods find use include:research applications, polypeptide synthesis applications andtherapeutic applications. Each of these representative categories ofapplications is described separately below in greater detail.

Research Applications

Examples of research applications in which the subject methods ofnucleic acid introduction find use include applications designed tocharacterize a particular gene. In such applications, the subject vectoris employed to introduce and express a gene of interest in a target celland the resultant effect of the inserted gene on the cell's phenotype isobserved. In this manner, information about the gene's activity and thenature of the product encoded thereby can be deduced. One can alsoemploy the subject methods to produce models in which overexpressionand/or misexpression of a gene of interest is produced in a cell and theeffects of this mutant expression pattern are observed.

Polypeptide Synthesis Applications

In addition to the above research applications, the subject methods alsofind use in the synthesis of polypeptides, e.g. proteins of interest. Insuch applications, a minimal plasmid vector that includes a geneencoding the polypeptide of interest in combination with requisiteand/or desired expression regulatory sequences, e.g. promoters, etc.,(i.e. an expression module) is introduced into the target cell, via invivo administration to the multicellular organism in which the targetcell resides, that is to serve as an expression host for expression ofthe polypeptide. Following in vivo administration, the multicellularorganism, and targeted host cell present therein, is then maintainedunder conditions sufficient for expression of the integrated gene. Theexpressed protein is then harvested, and purified where desired, usingany convenient protocol.

As such, the subject methods provide a means for at least enhancing theamount of a protein of interest in a multicellular organism. The term‘at least enhance’ includes situations where the methods are employed toincrease the amount of a protein in a multicellular organism where acertain initial amount of protein is present prior to in vivoadministration of the vector. The term ‘at least enhance’ also includesthose situations in which the multicellular organism includessubstantially none of the protein prior to administration of the vector.By “at least enhance” is meant that the amount of the particular proteinpresent in the host is increased by at least about 2 fold, usually by atleast about 5 fold and more usually by at least about 10 fold. As thesubject methods find use in at least enhancing the amount of a proteinpresent in a multicellular organism, they find use in a variety ofdifferent applications, including agricultural applications,pharmaceutical preparation applications, and the like, as well astherapeutic applications, described in greater detail infra.

Therapeutic Applications

The subject methods also find use in therapeutic applications, in whichthe vectors are employed to introduce a therapeutic nucleic acid, e.g.,gene, into a target cell, i.e., in gene therapy applications, to providefor persistent expression of the product encoded by the nucleic acidpresent on the vector. The subject vectors may be used to deliver a widevariety of therapeutic nucleic acids. Therapeutic nucleic acids ofinterest include genes that replace defective genes in the target hostcell, such as those responsible for genetic defect based diseasedconditions; genes which have therapeutic utility in the treatment ofcancer; and the like. Specific therapeutic genes for use in thetreatment of genetic defect based disease conditions include genesencoding the following products: factor VIII, factor IX, β-globin,low-density lipoprotein receptor, adenosine deaminase, purine nucleosidephosphorylase, sphingomyelinase, glucocerebrosidase, cystic fibrosistransmembrane conductor regulator, α1-antitrypsin, CD-18, ornithinetranscarbamylase, argininosuccinate synthetase, phenylalaninehydroxylase, branched-chain α-ketoacid dehydrogenase,fumarylacetoacetate hydrolase, glucose 6-phosphatase, α-L-fucosidase,β-glucuronidase, α-L-iduronidase, galactose 1-phosphateuridyltransferase, and the like, where the particular coding sequence ofthe above proteins that is employed will generally be the codingsequence that is found naturally in the host being treated, i.e., humancoding sequences are employed to treat human hosts. Cancer therapeuticgenes that may be delivered via the subject methods include: genes thatenhance the antitumor activity of lymphocytes, genes whose expressionproduct enhances the immunogenicity of tumor cells, tumor suppressorgenes, toxin genes, suicide genes, multiple-drug resistance genes,antisense sequences, and the like.

The subject methods also find use in the expression of RNA products,e.g., antisense RNA, ribozymes etc., as described in Lieber et al.,“Elimination of hepatitis C virus RNA in infected human hepatocytes byadenovirus-mediated expression of ribozymes,” J Virol. (1996 December)70(12):8782-91; Lieber et al., “Related Articles Adenovirus-mediatedexpression of ribozymes in mice,” J Virol. (1996 May) 70(5):3153-8; Tanget al., “Intravenous angiotensinogen antisense in AAV-based vectordecreases hypertension,” Am J Physiol. (1999 December) 277(6 Pt2):H2392-9; Horster et al. “Recombinant AAV-2 harboringgfp-antisense/ribozyme fusion sequences monitor transduction, geneexpression, and show anti-HIV-1 efficacy, Gene Ther. (1999 July)6(7):1231-8; and Phillips et al., “Prolonged reduction of high bloodpressure with an in vivo, nonpathogenic, adeno-associated viral vectordelivery of AT1-R mRNA antisense,” Hypertension. (1997 January) 29(1 Pt2):374-80. As such, the subject methods can be used to delivertherapeutic RNA molecules, e.g., antisense, ribozyme, etc., into targetcells of the host.

An important feature of the subject methods, as described supra, is thatthe subject methods may be used for in vivo gene therapy applications.By in vivo gene therapy applications is meant that the target cell orcells in which expression of the therapeutic gene is desired are notremoved from the host prior to contact with the vector system. Incontrast, the subject vectors are administered directly to themulticellular organism and are taken up by the target cells, followingwhich expression of the gene in the target cell occurs. Anotherimportant feature is that the resultant expression is persistent andoccurs without integration of the vector DNA into the target cellgenome.

Kits

Also provided by the subject invention are kits for use in practicingthe subject methods of nucleic acid delivery to target cells. Thesubject kits generally include the minicircle vector, which vector maybe present in an aqueous medium. The subject kits may further include anaqueous delivery vehicle, e.g. a buffered saline solution, etc. Inaddition, the kits may include one or more restriction endonucleases foruse in transferring a nucleic acid into the vector. In the subject kits,the above components may be combined into a single aqueous compositionfor delivery into the host or separate as different or disparatecompositions, e.g., in separate containers. Optionally, the kit mayfurther include a vascular delivery means for delivering the aqueouscomposition to the host, e.g. a syringe etc., where the delivery meansmay or may not be pre-loaded with the aqueous composition.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g. a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium, e.g.diskette, CD, etc., on which the information has been recorded. Yetanother means that may be present is a website address which may be usedvia the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

Methods of Minicircle Vector Production

As summarized above, also provided is a highly efficient method forproducing the subject minicircle vectors, as described above. Inproducing minicircle vectors according to this particular methodembodiment, a parent nucleic that includes an expression cassette ofinterest flanked by attBand attP sites of a unidirectional site specificrecombinase is contacted with the unidirectional site specificrecombinase that recognizes the flanking attB and attP sites underconditions sufficient for the unidirectional site specific recombinaseto mediate a recombination event that produces a minicircle vector fromthe parent nucleic acid, as described above. By “flanked” is meant thatthe expression cassette (or other sequence of interest that is to bepresent in the product minicircle vector, has an att site, e.g., attBand attP, at either end, such that the parent nucleic acid is describedby the formula:- - - - - - -att(P or B)-expression cassette-att(P or B)- - - - - - -The order of the att sites does not generally matter. The att sites aresubstrate sites for the unidirectioinal site specific recombinase, andare typically referred to as attB or attP sites by those of skill in theart. Sites of interest include, but are not limited to, the att sitesrecognized by the specific integrase recombinases above, as well asmutants thereof.

The parent nucleic acid may be present as a variety of different forms,depending at least in part on whether the production method is an invitro or in vivo method. As such, the parent nucleic acid may be alinear double stranded nucleic acid, a closed circular nucleic acid(such as a bacterial plasmid suitable for use in replication),integrated into genomic DNA, and the like.

As indicated above, the above method may be practiced in vitro or invivo, e.g., inside of a cell. Where the above method is practiced invitro, all necessary reagents, e.g., parent nucleic acid, site specificintegrase, etc., are combined into a reaction mixture and maintainedunder sufficient conditions for a sufficient period of time for the sitespecific recombinase mediated production of the desired productminicircle vectors to occur. Typically, for in vitro reactions, thereaction mixture is maintained at a temperature of between about 20 and40° C.

In certain embodiments, the above method is an in vitro method in thatthe recombinase mediated production of the desired product minicirclevector occurs inside of a cell in culture. Examples of such embodimentsincludes those embodiments where the parent nucleic acid is a plasmidthat replicated in a bacterial host to produce large copy numbers of theparent nucleic acid prior to the recombinase mediated vector productionstep.

In the above in vivo embodiments, the first step may generally be tofirst prepare a host cell that includes large numbers of the parentnucleic acid. This may conveniently be done by transforming a host cell,e.g., E. coli, with a plasmid that will serve as the parent nucleicacid. The resultant transformed host cell is then maintained underconditions sufficient for the host cell to produce large copy numbers ofthe parent nucleic acid, as described above.

Upon provision of the host cell having sufficient copy numbers of theparent nucleic acid (e.g., plasmid), the unidirectional site-specificrecombinase activity (i.e., that mediates production of the desiredvector from the parent nucleic acid) is then produced in the host cell.The desired recombinase activity may be produced in the cell using anyconvenient protocol. In certain embodiments, the recombinase or anucleic acid coding sequence therefore may be introduced into the hostcell, e.g., as described above. Alternatively, the coding sequence forthe recombinase may already be present in the host cell but notexpressed, e.g., because it is under the control of an induciblepromoter. In these embodiments, the inducible coding sequence may bepresent on the parent nucleic acid, present on another episomal nucleicacid, or even integrated into the host's genomic DNA. Representativeinducible promoters of interest that may be operationally linked to therecombinase coding sequence include, but are not limited to: aracBADpromoter, the lambda pL promoter, and the like. In these embodiments,the step of providing the desired recombinase activity in the host cellincludes inducing the inducible promoter to cause expression of thedesired recombinase.

Following production of the desired recombinase activity in the hostcell, the resultant host cell is then maintained under conditions andfor a period of time sufficient for the recombinase activity to mediateproduction of the desired minicircle vectors from the parent nucleicacids. Typically, the host cell is maintained at a temperature ofbetween about 20 and 40° C.

Following recombinase mediated production of the minicircle vectors fromthe parent nucleic acids, as described above, the product minicirclesmay then be separated from the remainder of their “synthesis”environment (e.g., reaction mixture, host cell, etc.) as desired. Anyconvenient protocol for separating the product minicircles may beemployed. Representative protocols are described in the experimentalsection below.

To assist in distinguishing/separating the desired product minicirclevectors from the byproduct circular remainder of the parent nucleicacid, the byproduct may be selectively cleaved to linearize thebyproduct. To provide for this selectable cleavage, a restriction site,e.g., IScel or other suitable site, may be provided in the parentnucleic acid that, following the recombinase mediated recombinationevent, is present in the byproduct, where the restriction site is thencleaved by its restriction endonuclease, which is provided in thereaction mixture or cell following production of the minicircles andparent byproduct. As with the provision of the recombinase activity,described above, the restriction endonuclease activity may be providedat the appropriate time by a number of different protocols, e.g., byintroducing the endonuclease or coding sequence therefor into thereaction mixture/cell following production of the parent byproduct, orinducing expression of the endonuclease coding sequence that is alreadypresent but not expressed in the reaction mixture or cell because it isunder the control of an inducible promoter, such as that describedabove. In these embodiments in which the vector production occurs in acell, the endonuclease is typically an endonuclease that is notendogenous to the host cell, where representative restrictionendonucleases of interest include, but are not limited to: IScel, I-CeuI, PI-Psp I and the like.

Also provided are systems for use in practicing the above-describedmethods of minicircle vector production. The subject systems typicallyat least include a parent nucleic or precursor thereof, e.g., a nucleicacid having att sites flanking a cloning site, and a host cell. Incertain embodiments, the parent nucleic acid further includes a codingsequence for a unidirectional site-specific recombinase that recognizesthe att sites, e.g., under the control of an inducible promoter, and/ora coding sequence for restriction endonuclease for cleaving a parentbyproduct, e.g., under the control of an inducible promoter. In theseembodiments, the system may also include an inducing agent, depending onthe nature of the inducible promoter. In yet other embodiments, thesystem may further include a separate source of the recombinase and/orrestriction endonuclease, as described above.

Also provided are kits for use in practicing the subject methods, wherethe kits may include one or more of the above components of the systems,e.g., parent nucleic acid, host cell, inducing agent, and the like. Inaddition to the above components, the subject kits will further includeinstructions for practicing the subject methods. These instructions maybe present in the subject kits in a variety of forms, one or more ofwhich may be present in the kit. One form in which these instructionsmay be present is as printed information on a suitable medium orsubstrate, e.g. a piece or pieces of paper on which the information isprinted, in the packaging of the kit, in a package insert, etc. Yetanother means would be a computer readable medium, e.g. diskette, CD,etc., on which the information has been recorded. Yet another means thatmay be present is a website address which may be used via the internetto access the information at a removed site. Any convenient means may bepresent in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

I. Materials and Methods

A. Vector Construction:

To prepare the hAAT minicircle construct pBAD.φC31.RHB (FIG. 1a ), weamplified φC31 integrase from the plasmid pCMV.φC31 (Groth, A. C.,Olivares, E. C., Thyagarajan, B. & Calos, M. P. A phage integrasedirects efficient site-specific integration in human cells. Proc NatlAcad Sci USA 97, 5995-6000. (2000)), using the following primers: 5′-CCGTCC ATG GAC ACG TAC GCG GGT GCT (SEQ ID NO:01), and 5′-ATG CGC GAG CTCGGT GTC TCG CTA CGC CGC TAC (SEQ ID NO:02), and inserted the PCR productinto Nco I and Sac I sites of pBAD/Myc-His (Invitrogen, Carlsbad,Calif.), resulting in an intermediate plasmid pBAD.φC31. We composedattB, and attP using the corresponding DNA oligonucleotides (Groth etal., supra), and inserted them into the Spe I and Kpn I sites,respectively, to flank the hAAT expression cassette of the plamsidpRSV.hAAT.bpA (FIG. 2a ). The attB, and attP binding sites andexpression cassette was inserted into Sac I and Kpn I sites ofpBAD.φC31, resulting in pBAD.φC31.RHB. We prepared the vectorpBAD.φC31.sApoE.hFIX (FIG. 1b ) to produce minicircle expressing hFIX byinserting the expression cassette, derived to frompBS.sApoE.HCR.hAAT.hFIX+IntA.bpA (Chen, Z. Y. et al. Linear DNAsconcatemerize in vivo and result in sustained transgene expression inmouse liver. Mol Ther 3, 403-410. (2001); Miao, C. H. et al. Inclusionof the hepatic locus control region, an intron, and untranslated regionincreases and stabilizes hepatic factor IX gene expression in vivo butnot in vitro. Mol Ther 1, 522-532 (2000); and Miao, C. H., Thompson, A.R., Loeb, K. & Ye, X. Long-term and therapeutic-level hepatic geneexpression of human factor IX after naked plasmid transfer in vivo. MolTher 3, 947-957. (2001)), into the Spe I site of pBAD.φC31.RHB after theexpression cassette was removed by Xho I digestion.

B. Production of Minicircles:

We used minicircle producing vectors to transform E. coli Top 10(Invitrogen, Calsbad, Calif.). The bacteria was grown using a NewBrunswick Scientific incubator (Model C24, Edison, N.J.). We obtainedL-(+)-arabinose from Sigma Chemical Co. (St. Lois, Mo.). We quantifiedthe DNA bands after agarose electrophoresis using Quant One of Bio-RedLaboratories (Hercules, Calif.). We prepared purified expressioncassette and 2-fragment DNA from plasmid pRSV.hAAT.bpA as describedpreviously (Chen et al., supra). We dialyzed all the DNA preparationsagainst TE overnight before delivery to animals.

C. Determination of Transgene Expression in Mice:

We obtained 6-8 week old C57BL/6 mice from Jackson Laboratory (BarHarbor, Me.). We delivered DNA to mouse livers using a hydrodynamictechnique (Zhang, G., Budker, V. & Wolff, J. A. High levels of foreigngene expression in hepatocytes after tail vein injections of nakedplasmid DNA. Hum Gene Ther10, 1735-1737 (1999); Liu, F., Song, Y. & Liu,D. Hydrodynamics-based transformation in animals by systemicadministration of plasmid DNA. Gene Ther 6, 1258-1266 (1999)). Wecollected mouse blood periodically using a retro-orbital procedure, anddetermined serum hAAT and hFIX by ELISA as described earlier (Yant, S.R. et al. Somatic integration and long-term transgene expression innormal and haemophilic mice using a DNA transposon system. Nat Genet 25,35-41. (2000)). All animals were treated under the NIH and StanfordUniversity Animal Care Guidelines.

D. Southern Blot Analysis of Vector DNA Structure in Mouse Livers:

We prepared liver DNA using a salt out procedure. Twenty μg of liver DNAfrom mice receiving one of the four forms of vector DNA expressing hAATwas digested with Bgl II, which did not cut the vector, or Hind III,which cut once through the expression cassette. We separated therestricted DNAs by electrophoresis in a 0.8% agarose gel, and blotted itonto a nitrocellular membrane. Vector DNA was detected afterhybridization with a p32-dCTP-labelled hAAT cDNA probe, andautoradiography or Phosphoimager.

II. Results

A. Production of Minicircles

We constructed the plasmids pBAD.φC31.RHB (FIG. 1a ) and pBAD.φC31.hFIX(FIG. 1b ) as precursors to the production of minicircular vectorsexpressing human al-antitrypsin (hAAT), and human factor IX (hFIX),respectively.

To determine the optimal conditions for induction of φC31integrase-mediated recombination, an overnight culture (OD₆₀₀˜2.50)obtained from a single colony of transformed cells containingpBAD.φC31.RHB was prepared. We determined that the optimal incubationtemperature was 32° C., with 1% inducer L-(+)-arabinose added to thebacterial culture (data not shown). However, we found that therecombination efficiency was poor when induction was carried out byadding L-(+)-arabinose to the overnight bacterial growth (FIGS. 1c, and1d ). The recombination efficiency was greatly enhanced by re-suspendingthe bacteria in fresh LB broth before adding L-(+)-arabinose. Moreover,a slightly better yield of minicircle was obtained when the bacterialculture was re-suspended at a 4 to 1 ratio of overnight growth volume vsfresh LB volume compared with a 1 to 1 ratio (FIGS. 1c and 1d ). Theoptimal conditions for minicircle production include resuspending theovernight bacterial growth 4:1 in fresh LB broth containing 1%L-(+)-arabinose, and incubating the bacteria at 32° C. with shaking at250 rpm for 60 to 120 minutes. Because the minicircle was about aquarter of the size of the parent vector pBAD.φC31.RHB, we estimatedthat under these culture conditions, the efficiency of recombination wasgreater than 97 percent (FIG. 1d ).

We purified recombined DNAs from bacteria growth using Qiagen plasmidDNA Kit, and purified minicircles by standard gradient CsCl bandingprocedure (Sambrook, J., Fritsch, E. F. & Maniatis, T. (eds.). Molecularcloning: a laboratory manual, (Cold Spring Harbor Laboratory, New York,1989)) after linearizing the bacteria backbone circle with Nco Idigestion. We obtained about 1 to 1.5 mg of recombined DNA before CsClpurification and 150 to 200 μg of purified minicircle from 1,000 ml ofbacteria growth with minicircle-producing vector pBAD.φC31.RHB, orpBAD.φC31.hFIX.

B. Minicircle-Mediated Transgene Expression In Vivo

To determine if the hAAT-expressing minicircle was devoid of bacterialDNA silencing in vivo, we compared the expression profiles of thisminicircular DNA with equal molar amounts of un-recombined plasmidpRSV.hAAT.bpA (FIG. 2a ), a linear DNA mixture of expression cassetteand bacterial backbone, or equal molar amounts of purified linearexpression cassette containing the same DNA sequence as the minicircleexcept for the 37 bp attR hybrid site after transformation into mouseliver.

Consistent with our previous observation (Chen et al., manuscriptsubmitted), serum concentrations of hAAT obtained from mice injectedwith purified expression cassette was more than 3-fold higher than thatof mice received 2-fragment DNA, and 20- to 43-fold higher than ccDNAinjected mice (FIG. 2b , left panel) 3 weeks after DNA infusion. Themice receiving minicircle DNA produced 10- to 13-fold more serum hAATthan those receiving the purified expression cassette, which was 200- to560-fold higher than that of ccDNA group. Mice receiving ccDNA alsoexpressed to a high level of serum hAAT initially, but the serumreporter level dropped by 710-fold in the first 3 weeks, and continuedto decrease afterward. Our data clearly demonstrate that the minicirclewas the most efficient vector form and could express persistent and highlevels of transgene product.

To demonstrate the potential for therapeutic efficacy, we compared thehFIX expressing minicircle to the corresponding un-recombined plasmid.Animals infused with this minicircle expressed a high level of serumhFIX which stabilized at about 12 microgram hFIX per ml of serum (morethan twice normal) for up to 7 weeks (length of experiment. FIG. 2b ,right panel). High levels of serum hFIX were obtained in mice receivingthe un-recombined plasmid one day after DNA infusion, but the serumtherapeutic protein dropped more than 45-fold in 3 weeks and continuedto decrease afterward.

C. Southern Blot Analysis of Minicircle DNA in Mouse Livers

Although in previous studies, we found no difference in the amount ofDNA after infusion of ccDNA or linear DNA (Chen et al., supra), wewanted to establish if the same was true in minicircle injected mice.Liver vector DNA copy number was determined in mice receiving differentforms of the hAAT vector DNA 15 weeks after injection (FIG. 2b , leftpanel). About 13 to 20 copies of vector DNA per diploidy mouse genomewas detected in each group (FIG. 3a ). Consistent with previousobservations, our data indicate that difference in serum hAAT levels wasnot due to variations in the amount of vector DNA in mouse liver.

Previously, we have demonstrated that circular plasmids remained asintact circles in mouse liver (Chen et al., supra) (Chen et al.,manuscript submitted). In order to establish if minicircle DNA behavedlike other circular plasmids in mouse livers, we analyzed the molecularstructure of vector DNA by Southern blot. With Bgl II digestion, whichdoes not cut in the vector, we found multiple bands representingaggregates of supercoiled minicircles. These bands were converted into asingle length monomer by digestion with Hind III, which cuts once in thevector (FIG. 3b ). Thus, similar to the uncut circular plasmid, theminicircle DNA was maintained as an intact episomal circle in mouseliver. In addition, consistent with our previous observations, the 2linearized DNAs formed large concatemers, as represented by the 23 kbbands, as well as small circles (FIG. 3b ).

III. Discussion

We demonstrate above that large quantities of minicircle DNA vectorsdevoid of bacterial DNA sequences can be produced by using the phageφ031 integrase-mediated recombination in E. coli. The technique isrelatively simple, the yield is high, and the production can be easilyscaled up. We establish that minicircles can express high and persistentlevels of transgene products in mouse liver. Minicircles expressed 45-and 560-fold more serum hFIX and hAAT than their parent unrecombinedplasmids in mouse liver. Importantly, and similar to our previousresults (Chen et al., supra) (Chen et al., manuscript submitted), thisdifference in gene expression was not related to changes in the amountof vector DNA in mouse liver. Together, these results further confirmthe finding that the bacterial backbone plays an inhibitory role inepisomal transgene expression. As compared to the linear purifiedexpression cassette, minicircle DNA expressed more than 10-fold higherlevels of serum hAAT, suggesting that the minicircle was an optimalepisomal vector form for transgene expression, probably because of itscircular configuration. Alternatively, substantial amounts of linearexpression cassette might be inactivated via the partial loss ofpromoter, and/or polyadenylation DNA sequences during thenon-homology-end-joining process.

Since the transcriptional silencing effect is overcome by usingminicircular DNA, transgene expression will not be lost except duringcell division or cell death. It has been hypothesized that when plasmidDNA is delivered within some lipid DNA complexes, a loss of transgeneexpression occurs due to an immune response against CpG dinucleotidespresent in bacterial DNA. We have previously established that this isunlikely to occur in our studies because there is no loss of DNA andsimilar expression profiles are found in normal and immunodeficient mice(Chen et al., supra) (Chen et al., manuscript submitted). It has beenwell documented that plasmids can undergo nucleation, and persist in anepisomal status for months or years not only in liver (Chen et al.,supra), but also in heart (Gal, D. et al. Direct myocardialtransformation in two animal models. Evaluation of parameters affectinggene expression and percutaneous gene delivery. Lab Invest 68, 18-25.(1993)), and skeletal muscle (Wolff, J. A., Ludtke, J. J., Acsadi, G.,Williams, P. & Jani, A. Long-term persistence of plasmid DNA and foreigngene expression in mouse muscle. Hum Mol Genet 1, 363-369. (1992)). Itis reasonable to expect that persistence of transgene expression fromminicircle can also be achieved from these, and other organs, with a lowcell turnover rate.

IV. One Step Column Purification of Minicircle DNA Vector from BacteriaGrowth

A. Abstract:

The following discussion demonstrations the use of a one-step columnpurification protocol of minicircle DNA vector from bacterial growththat produces about 1 mg of minicircle DNA vector with more than 96%purity from 1,000 ml of bacterial growth using Qiagen DNA Kit withoutadditional work. The following protocol enables production of largequantity of minicircle vector for clinical use.

B. Plasmid Construct:

FIG. 4a schematically illustrates the minicircle producing constructp2×BAD.φC31.hFIX.Isce Ig+s for this study. Two copies of the integraseφ031 gene, and one copy of the restriction enzyme Isce I gene are allplaced under the control of araC/BAD promoter. The expression cassettesApoE.hFIX.bpA flanked with attB and attP, and an I-Sce I restrictionsite (I-Sce Is) are included in the same construct. The plasmid backboneis pUC19 containing a pUC DNA replication origin (UC), and an ampicillinresistance gene (Amp^(R)).

C. Preparation of Minicircle:

The plasmid p2×(BAD.φC31).hFIX.Isce Ig+s (FIG. 4a ) was used totransform Top 10 bacteria. A colony of the transformed bacteria wasgrown overnight in 200 ml of LB broth with 10 mg/ml of ampicillin usinga standard bacteria growth procedure. The bacteria was further grownovernight in 1500 ml of LB/ampicillin broth. The bacteria was spun down,re-suspended 4 to 1 in fresh LB broth containing 1% of L-arabinose,incubated at 32° C. with constant shaking at 250 rpm for one hour asdescribed above. One half of the bacteria were then incubatedcontinuously at 32° C. for additional two hours, while another half wereincubated at 37° C. for 2 hours. The bacteria was spun down, andprocessed for plasmid DNA preparation using Qiagen kit.

FIG. 5 demonstrates the purity of the minicircle vector DNA. 0.8 μg ofDNA each was digested with BgI II and Eco N1, each of them cuts oncethrough the expression cassette or the bacterial backbone, respectively.The 1.4 kb and 12.3 kb bands in lane 1 were the restriction products ofunrecombined plasmid (FIG. 4a ), while the 4.1 kb and the band slightlybelow 12.3 kb in lane 2 and 3 from above-mentioned 2 differentrecombination-restriction conditions were the linearized minicircle(FIG. 4b ) and bacterial DNA (FIG. 4c ), respectively.

Quantification of the DNA bands (Quant One of Bio-Rad, Hercules, Calif.)demonstrated that the purity of the minicircle in lane 2 and 3 were 96%and 97% respectively.

D. Discussion:

The technology of one step column purification of minicircle DNA vectorfrom bacteria growth enables large scale production of minicircle forclinical use. This technology saves time and materials so that is morecost-effective. More importantly, it allows production of largequantities of minicircle vector for clinical use without involving anytoxic material, such as ethium bromide. In this one step protocol, theI-Sce I gene is included to express the restriction enzyme whichlinearizes the circular DNA by cutting a built-in I-Sce I restrictionsite in the minicircle producing construct. The linearized DNA is thendegraded by the bacterial nucleases, and the minicircle will become theonly episomal DNA to be purified by commercially available kit.Currently, both φC31 and I-Sce I genes are driven by araCBAD promoter aothat the two enzymes are induced simultaneously upon addition of theinducer L-arabinose. Consequently, circular bacterial DNA linearizationand the φC31-mediated minicircle formation occur at the same time,resulting in a partial loss of unrecombined plasmid and hence a loweryield of minicircle DNA. However, we reason that percent of prematurelinearization/degradation is limited, because the recombinase φC31 ishighly efficient and relatively stable. The φC31 can process therecombination reaction to almost completeness in 30 to 60 minutes. Incontrast, the I-Sce I enzyme is unstable with a half-life of only 5minutes also. The I-Sce I enzyme in the bacteria could not reach a highlevel. Furthermore, we use 2 copies of the φC31 recombinase to furtherspeed up the minicircle formation and decrease the prematurelinearization/degradation further.

The above-described invention is the product of highly unexpectedresults observed by the inventors. Specifically, prior to the inventors'work described herein, it was believed, and also observed, that circularvectors, e.g., plasmids, could not provide for persistent high-levelprotein expression. As reported herein, by removing bacterial sequencesfrom circular vectors, the inventors were unexpectedly able to obtaincircular vectors that provide for persistently high levels of proteinexpression. Based on knowledge of the inventors at the time of filing ofthe present application, it was not at all obvious that one couldachieve persistent high-level expression from circular vectors byremoving bacterial silencing sequences from the vectors.

It is evident from the above results and discussion that an improvedmethod of transferring a nucleic acid into a target cell is provided bythe subject invention. Specifically, the subject invention provides ahighly efficient transgene expression vector which does not employ viralvectors and does not require target cell genome integration and yetprovides for persistent high level gene expression and thereforeprovides many advantages over prior art methods of nucleic acidtransfer. Also provided is a highly efficient and readily scalablemethod for producing the vectors employed in the subject methods. Assuch, the subject invention represents a significant contribution to theart.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A bacterial host cell comprising: (i) a parentnucleic acid that comprises: (a) a cloning site flanked by sites thatare recognized by a unidirectional site specific-recombinase, and (b) anexpression silencing bacterial DNA sequence, wherein the sites that arerecognized by the unidirectional site specific recombinase arepositioned between the cloning site and the expression silencingbacterial DNA sequence; and (ii) a nucleic acid comprising a codingsequence for said unidirectional site-specific recombinase.
 2. The hostcell according to claim 1, wherein said cloning site is part of amultiple cloning site.
 3. The host cell according to claim 1, whereinsaid cloning site comprises an expression cassette.
 4. The host cellaccording to claim 1, wherein said coding sequence for saidunidirectional site-specific recombinase is operably linked to aninducible promoter.
 5. The host cell according to claim 1, wherein: (1)said parent nucleic acid further comprises a restriction endonucleasesite recognized by a restriction endonuclease; and (2) said host cellfurther comprises a nucleic acid that comprises a coding sequence forsaid restriction endonuclease.
 6. The host cell according to claim 5,wherein said coding sequence for said restriction endonuclease isoperably linked to an inducible promoter.
 7. The host cell according toclaim 5, wherein the nucleic acid that comprises the coding sequence forsaid restriction endonuclease is not integrated into the host cell'sgenome.
 8. The host cell according to claim 5, wherein the nucleic acidthat comprises the coding sequence for said restriction endonuclease isintegrated into the host cell's genome.
 9. The host cell according toclaim 1, wherein said parent nucleic acid is integrated into said hostcell's genome.
 10. The host cell according to claim 1, wherein saidparent nucleic acid is not integrated into said host cell's genome. 11.The host cell according to claim 10, wherein said parent nucleic acid iscircular.
 12. The host cell according to claim 1, wherein the nucleicacid comprising said coding sequence for said unidirectionalsite-specific recombinase is integrated into the host cell's genome. 13.The host cell according to claim 12, wherein the coding sequence for theunidirectional site specific recombinase is operably linked to aninducible promoter.
 14. The host cell according to claim 1, wherein saidunidirectional site-specific recombinase is selected from the groupconsisting of φC31, R4, TP901-1, A118, and φFC1.
 15. The host cellaccording to claim 1, wherein said unidirectional site-specificrecombinase is φC31.
 16. The host cell according to claim 1, whereinsaid parent nucleic acid and said nucleic acid comprising the codingsequence for the unidirectional site-specific recombinase are part ofthe same nucleic acid molecule.
 17. The host cell according to claim 16,wherein said same nucleic acid molecule is not integrated into the hostcell's genome.